Method of preparing a guanosine-group compound

ABSTRACT

A glyoxal-guanosine-group compound is prepared either by reacting glyoxal-guanine with any one of ribose-1-phosphate and 2-deoxyribose-1-phosphate in the presence of purine nucleoside phosphorylase, or by reacting glyoxal-guanine with any one selected from the group consisting of uridine, 2&#39;-deoxyuridine and thymidine, together with phosphate ion, in the presence of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase. The glyoxal-guanosine-group compound is then decomposed by alkali, whereby a guanosine-group compound consisting of guanosine and 2&#39;-deoxyguanosine is prepared.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromthe prior Japanese Patent Application No. 2000-087302, filed Mar. 27,2000, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a method of preparing a guanosine-groupcompound, which is used as a raw material of an anti-virus agent,antisense medicine and the like, and a method of preparing aglyoxal-guanosine-group compound as an intermediate of theguanosine-group compound.

Guanosine or 2′-deoxyguanosine is industrially produced mainly byextraction/separation of hydrolysate of DNA (deoxyribonucleic acid) orRNA (ribonucleic acid), because the yield is extremely low whenguanosine or 2′-deoxyguanosine is chemically synthesized. However, thehydrolysate of DNA contains 2′-deoxyadenosine, 2′-deoxycytidine andthymidine other than the targeted 2′-deoxyguanosine. The hydrolysate ofRNA contains adenosine, cytidine and uridine other than the targetedguanosine. Accordingly, in order to collect only guanosine or2′-deoxyguanosine, it is necessary to perform complicated processes ofextraction/separation, thereby inevitably raising the production cost.

On the other hand, a method has been reported in which nucleoside (ordeoxynucleoside) and nucleic acid base, which are the raw materials, aresubjected to a base-exchange reaction by nucleoside phosphorylase,whereby the aimed nucleoside (or the aimed deoxynucleoside) is obtained(Hori, N., Watanabe, M., Yamazaki, Y., Mikami, Y., Agric. Biol. Chem.,53, 197-202 (1989)). By this method, adenosine and 2′-deoxyadenosine canbe easily prepared (Jpn. Pat. Appln. KOKAI Publication No. 11-46790“Method of preparing a purine nucleoside compound”). In order to obtainguanosine and 2′-deoxyguanosine by this method of enzymatic synthesisreaction, guanine must be soluble to water at least in a range of pHwhere the enzymes necessary for the reaction can function. In general,it is considered that the solubility of the reaction substrate ispreferably at least equal to the concentration of Michaelis constant(Km) of the enzyme or higher, in order to carry out the enzyme reactionsmoothly. However, as the solubility of guanine to water does not exceeda few ppm, the above method of enzymatic synthesis reaction cannot beemployed in practice. Due to such an impasse-like situation, it has beendemanded to develop a method of efficiently preparing guanosine or2′-deoxyguanosine.

BRIEF SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of efficientlypreparing guanosine or 2′-deoxyguanosine (i.e., a guanosine-groupcompound) at a high yield, as well as a method of efficiently preparinga glyoxal-guanosine or glyoxal-2′-deoxyguanosine (i.e.,glyoxal-guanosine-group compound) as an intermediate of theguanosine-group compound at a high yield.

The inventors of the present invention have assiduously studied forsolving the aforementioned problems. As a result, the inventors havediscovered the following 1)-6).

1) When guanine, whose solubility to water does not exceed a few ppm, isreacted with glyoxal, glyoxal-guanine represented by the followingformula (1) is obtained, and glyoxal-guanine is soluble to water.

(Glyoxal-guanine is also referred to as6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one.)

2) When microorganism itself which contains purine nucleosidephosphorylase (EC 2.4.2.1) or this enzyme derived from the microorganismare applied, for a reaction, to glyoxal-guanine and any one ofribose-1-phosphate and 2-deoxyribose-1-phosphate, aglyoxal-guanosine-group compound (i.e., glyoxal-guanosine orglyoxal-2′-deoxyguanosine) represented by the following formula (2) isobtained. Alternatively, when microorganism itself which contains purinenucleoside phosphorylase (EC 2.4.2.1) and pyrimidine nucleosidephosphorylase (EC 2.4.2.2) or these enzymes derived from themicroorganism are applied, for a reaction, to glyoxal-guanine and anyone selected from the group consisting of uridine, 2′-deoxyuridine andthymidine under the presence of phosphate ion, a glyoxal-guanosine-groupcompound (i.e., glyoxal-guanosine or glyoxal-2′-deoxyguanosine)represented by the following formula (2) is obtained. In other words,glyoxal-guanine can be a substrate of purine nucleoside phosphorylase.

(wherein R represents a hydrogen atom or a hydroxyl group.)

(Glyoxal-guanosine is also referred to as3-(β-D-erythropentofuranosyl)-6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one,and glyoxal-2′-deoxyguanosine is also referred to as3-(2-deoxy-β-D-erythropentofuranosyl)-6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one.)

3) In the method of preparing a glyoxal-guanosine-group compounddescribed in 2) above, when at least one compound selected from thegroup consisting of glycine, iminodiacetic acid, nitrilotriacetic acid,ethylenediaminetetraacetic acid (which will be referred to as “EDTA”hereinafter), ethylene glycol bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (which will be referred to as “EGTA”hereinafter) and salts of these substances is added, or the above atleast one compound is added in combination with boric acid or a saltthereof, the reaction yield is significantly improved. The abovecompound is added as a stabilizer of ribose-1-phosphate or2-deoxyribose-1-phosphate, which is the substrate or an intermediateproduced during base-exchange reaction.

4) As a principle, nucleoside phosphorylase derived from variousorganism can be efficient as the enzyme to be used in the preparationmethod of 2) above. However, the enzyme derived from microorganism ofBacillus genus, Escherichia genus or Klebsiella genus is suitable, andthe thermotolerant enzyme derived from Bacillus stearothermophilus JTS859 (FERM BP-6885) is especially preferable.

5) When an alkali is applied, for a reaction, to theglyoxal-guanosine-group compound obtained by the method of 2) above, theglyoxal portion of the glyoxal-guanosine-group compound is easilyreleased, whereby a guanosine-group compound (i.e., guanosine or2′-deoxyguanosine) can be obtained.

6) The preparation processes of the aforementioned 1)-5) can be carriedout by one-pot reaction.

On the basis of the aforementioned discoveries, the present inventionhas been completed.

In summary, the present invention relates to the following methods(1)-(16).

(1) A method of preparing a glyoxal-guanosine-group compound representedby the formula (2):

wherein R represents a hydrogen atom or a hydroxyl group, whichcomprises the step of:

reacting glyoxal-guanine represented by the formula (1):

 with ribose-1-phosphate or 2-deoxyribose-1-phosphate in the presence ofpurine nucleoside phosphorylase, thereby obtaining a glyoxal-guanosineor glyoxal-2′-deoxyguanosine.

(2) A method of preparing a glyoxal-guanosine-group compound representedby the formula (2):

wherein R represents a hydrogen atom or a hydroxyl group, whichcomprises the step of:

reacting glyoxal-guanine represented by the formula (1):

 with any one selected from the group consisting of uridine,2′-deoxyuridine and thymidine, together with phosphate ion, in thepresence of purine nucleoside phosphorylase and pyrimidine nucleosidephosphorylase, thereby obtaining a glyoxal-guanosine orglyoxal-2′-deoxyguanosine.

(3) A method of preparing a guanosine-group compound, which comprisesthe steps of:

reacting glyoxal-guanine represented by the formula (1):

 with ribose-1-phosphate or 2-deoxyribose-1-phosphate in the presence ofpurine nucleoside phosphorylase, thereby obtaining a compoundrepresented by the formula (2):

 wherein R represents a hydrogen atom or a hydroxyl group; and

decomposing, by alkali, the compound represented by the formula (2),thereby obtaining guanosine or 2′-deoxyguanosine.

(4) A method of preparing a guanosine-group compound, which comprisesthe steps of:

reacting glyoxal-guanine represented by the formula (1):

 with any one selected from the group consisting of uridine,2′-deoxyuridine and thymidine, together with phosphate ion, in thepresence of purine nucleoside phosphorylase and pyrimidine nucleosidephosphorylase, thereby obtaining a compound represented by the formula(2):

 wherein R represents a hydrogen atom or a hydroxyl group; and

decomposing, by alkali, the compound represented by the formula (2),thereby obtaining guanosine or 2′-deoxyguanosine.

(5) A method of preparing a glyoxal-guanosine-group compound describedin the aforementioned (1), wherein, as purine nucleoside phosphorylase,a microorganism itself which contains the enzyme or the enzyme derivedfrom the microorganism is used.

(6) A method of preparing a glyoxal-guanosine-group compound describedin the aforementioned (2), wherein, as purine nucleoside phosphorylaseand pyrimidine nucleoside phosphorylase, a microorganism itself whichcontains the enzymes or the enzymes derived from the microorganism areused.

(7) A method of preparing a guanosine-group compound described in theaforementioned (3), wherein, as purine nucleoside phosphorylase, amicroorganism itself which contains the enzyme or the enzyme derivedfrom the microorganism is used.

(8) A method of preparing a guanosine-group compound described in theaforementioned (4), wherein, as purine nucleoside phosphorylase andpyrimidine nucleoside phosphorylase, a microorganism itself whichcontains the enzymes or the enzymes derived from the microorganism areused.

(9) A method of preparing a glyoxal-guanosine-group compound describedin the aforementioned (5) or (6), wherein the microorganism belongs toBacillus genus, Escherichia genus or Klebsiella genus.

(10) A method of preparing a guanosine-group compound described in theaforementioned (7) or (8), wherein the microorganism belongs to Bacillusgenus, Escherichia genus or Klebsiella genus.

(11) A method of preparing a glyoxal-guanosine-group compound describedin any one of the aforementioned (5), (6) and (9), wherein themicroorganism is Bacillus stearothermophilus JTS 859 (FERM BP-6885),Escherichia coli IFO 3301, Escherichia coli IFO 13168, or Klebsiellapneumoniae IFO 3321.

(12) A method of preparing a guanosine-group compound described in anyone of the aforementioned (7), (8) and (10), wherein the microorganismis Bacillus stearothermophilus JTS 859 (FERM BP-6885), Escherichia coliIFO 3301, Escherichia coli IFO 13168, or Klebsiella pneumoniae IFO 3321.

(13) A method of preparing a glyoxal-guanosine-group compound describedin any one of the aforementioned (1), (2), (5), (6), (9) and (11),wherein at least one compound selected from the group consisting ofglycine, iminodiacetic acid, nitrilotriacetic acid,ethylenediaminetetraacetic acid, ethylene glycol bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid and salts thereof is added, or theabove at least one compound is added in combination with boric acid or asalt thereof.

(14) A method of preparing a guanosine-group compound described in anyone of the aforementioned (3), (4), (7), (8), (10) and (12), wherein atleast one compound selected from the group consisting of glycine,iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraaceticacid, ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acidand salts thereof is added, or the above at least one compound is addedin combination with boric acid or a salt thereof.

(15) A method of preparing a glyoxal-guanosine-group compound describedin any one of the aforementioned (1), (2), (5), (6), (9), (11) and (13),which is performed by one-pot reaction.

(16) A method of preparing a guanosine-group compound described in anyone of the aforementioned (3), (4), (7), (8), (10), (12) and (14), whichis performed by one-pot reaction.

Additional objects and advantages of the invention will be set forth inthe description which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and obtained by means ofthe instrumentalities and combinations particularly pointed outhereinafter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described in detail hereinafter.

<Raw Material>

Glyoxal-guanine (i.e.,6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one) to be used as araw material in the present invention can be prepared at a high yield byadding guanine to a glyoxal aqueous solution and stirring the mixturewith heating at a high temperature (preferably at 50-75° C.) for about15-20 hours. The solubility of glyoxal-guanine to water increases as thetemperature rises. After the completion of the reaction, glyoxal-guanineis crystallized by cooling and can be obtained as a white solid byfiltering.

However, when one-pot reaction is carried out by continuously addingnucleoside phosphorylase and either ribose donor or 2-deoxyribose donor,the solution in which glyoxal-guanine has been obtained may be used, asit is, as a raw material solution.

In the present invention, ribose-1-phosphate and uridine used as theribose donor, and 2-deoxyribose-1-phosphate, 2′-deoxyuridine andthymidine used as the 2-deoxyribose donor are commercially available by,for example, Sigma Aldrich Japan Co.

<Synthesis Conditions of a Glyoxal-guanosine-group Compound by theEnzyme Reaction>

The ribose donor or the 2-deoxyribose donor (which will be referred toas “the ribose-group donor” hereinafter) and glyoxal-guanine are addedto a phosphate buffer solution (pH 6-8, preferably pH 7). Microorganismitself or the enzymes derived from the microorganism, which producespurine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase(or only purine nucleoside phosphorylase), is added to the abovephosphate buffer mixture. The resultant reaction mixture is stirred atthe optimum temperature for the enzyme for 24-60 hours, resulting inreactions as represented by “Reaction formula 1 and Reaction formula 2”or “Reaction formula 3 and Reaction formula 4”.Glyoxal-2′-deoxyguanosine is obtained by “Reaction formula 1 andReaction formula 2”. Glyoxal-guanosine is obtained by “Reaction formula3 and Reaction formula 4”. In Reaction formula 1 and Reaction formula 3as the former half part, pyrimidine nucleoside phosphorylase is used asthe enzyme in the reaction. In Reaction formula 2 and Reaction formula 4as the latter half part, purine nucleoside phosphorylase is used as theenzyme in the reaction. With respect to the optimum temperature for theenzyme, the temperature may be in the range of approximately 40-70° C.in the case of the enzyme derived from Bacillus stearothermophilus JTS859 (FERM BP-6885).

In the enzyme reaction of the present invention, purine nucleosidephosphorylase and pyrimidine nucleoside phosphorylase (or only purinenucleoside phosphorylase) are used. When pyrimidine nucleoside (uridine,2′-deoxyuridine, or thymidine) is used as the ribose-group donor,pyrimidine nucleoside phosphorylase is essentially required forproducing ribose-1-phosphate or 2-deoxyribose-1-phosphate in “Reactionformula 1” or “Reaction formula 3”, as the former half reaction. On theother hand, when ribose-1-phosphate or 2-deoxyribose-1-phosphate is usedas the ribose-group donor from the start of the reaction, the reactionrepresented by “Reaction formula 1” or “Reaction formula 3”, is notnecessitated, and thus pyrimidine nucleoside phosphorylase is notrequired. However, in this case, even if pyrimidine nucleosidephosphorylase is present, the overall enzyme reaction will not beadversely affected at all.

In Reaction formulae 1-4, “PYNP” represents pyrimidine nucleosidephosphorylase, and “PUNP” represents purine nucleoside phosphorylase.

In the present invention, the initial concentration of the ribose-groupdonor as the raw material is 5-1000 mM, preferably 10-100 mM.Glyoxal-guanine does not dissolve in a large amount at a time. However,if it is assumed that the initially added amount of glyoxal-guanine wereto be completely dissolved at a time, the expressed initialconcentration would be 5-1000 mM, preferably 10-100 mM. The initialconcentration of phosphate ion of 1-20 mM suffices the purpose.

<Stabilizer of Deoxyribose-1-phosphate, etc.>

In the present invention, even when a nucleoside (uridine,2′-deoxyuridine, thymidine) is used as the raw material, the reactionproceeds by way of ribose-1-phosphate or 2-deoxyribose-1-phosphate asthe reaction intermediate, as shown in the aforementioned formulae.These phosphate compounds are unstable by nature and, when being left,naturally decomposed into ribose and phosphate ion or 2-deoxyribose andphosphate ion as time passes. When phosphatase is present in thereaction system, the decomposition is accelerated. If such decompositionoccurs, the production efficiency of glyoxal-guanosine-group compound isdecreased, because ribose or 2-deoxyribose, which is the product bydecomposition of the intermediate, does not serve as the substrate ofnucleoside phosphorylase. Therefore, in order to increase the productionefficiency, a substance, which stabilizes ribose-1-phosphate or2-deoxyribose-1-phosphate and inhibits the activity of phosphatase, hasbeen searched in the present invention.

As a result, it has been discovered that addition of at least onecompound selected from the group consisting of glycine, iminodiaceticacid, nitrilotriacetic acid, EDTA, EGTA and salts thereof, and morepreferably, addition of the above at least one compound in combinationwith boric acid or a salt thereof is effective. The combination of EDTAor a salt thereof with boric acid or a salt thereof exhibits asignificantly excellent effect. The preferable concentration of eachcomposition added is 20-200 mM for boric acid or a salt thereof, and2-10 mM for the above at least one compound or salts thereof (i.e.,glycine, iminodiacetic acid, nitrilotriacetic acid, EDTA, EGTA). Saltsof the above compounds and a salt of boric acid used herein are notparticularly limited, and any salts can be used. For example, the saltsused herein may be salts of alkaline metal such as sodium and potassium,or salts of alkaline earth metal such as calcium and magnesium.

<Preparation of a Guanosine-group Compound from aGlyoxal-guanosine-group Compound>

A glyoxal-guanosine-group compound releases, by decomposition, theglyoxal portion thereof in an alkali aqueous solution, thereby obtaininga guanosine-group compound. As the alkali aqueous solution, 0.05-0.5N,preferably 0.1-0.25N sodium hydroxide aqueous solution can be used, forexample. This decomposition reaction is accelerated by the increase inpH and temperature. However, it is essential to select a mild conditionin which the bonding with ribose-group is not broken. Specifically, aglyoxal-guanosine-group compound is relatively stable in the range of pH4-8, but is decomposed and releases the glyoxal portion thereof at pH 9or higher. In conclusion, it is preferable that aglyoxal-guanosine-group compound is decomposed at 60-80° C. in the rangeof pH 9-11 in 1-5 hours duration, to obtain a guanosine-group compound.

<Separation and Purification of a Guanosine-group Compound>

Collection of the product from the reaction solution can be performed byultrafiltration, ion exchange separation, adsorption chromatography,crystallization and the like. The amount of the reaction product can bedetermined by the HPLC method using a UV detector.

<Microorganism and Enzyme>

Purine nucleoside phosphorylase (EC 2.4.2.1) and pyrimidine nucleosidephosphorylase (EC 2.4.2.2) used in the present invention may have anyorigin as a principle. The enzymes produced by the microorganismdescribed below are preferable. The microorganism to be used in thepresent invention is not particularly limited to such examples, as longas the microorganism produces the aforementioned enzymes in asignificant amount.

However, as shown in the aforementioned Reaction formulae, the presentreactions employ nucleoside or 2′-deoxynucleoside as the substrate andthus inevitably produce a phosphate compound as the intermediate.Accordingly, microorganism which shows a strong nucleosidase and/orphosphatase activity cannot be utilized.

Examples of the microorganism which suffices these conditions includethose which belong to Bacillus genus, Escherichia genus or Klebsiellagenus. More specifically, the examples include Bacillusstearothermophilus JTS 859 (FERM BP-6885), Escherichia coli IFO 3301,Escherichia coli IFO 13168, and Klebsiella pneumoniae IFO 3321.

In the present reaction, the higher the solubility and the dissolvingrate of the raw material are, the higher yield and the higher reactionrate are obtained. Accordingly, a relatively high temperature during thereaction is advantageous. Due to this, use of thermophilic bacteria andheat-resistant enzymes is preferable. Among the aforementioned examplesof microorganisms, Bacillus stearothermophilus JTS 859 is the mostpreferable.

“IFO” indicates that the microorganisms are preserved in Institute forFermentation, Osaka (IFO), Feb. 2, 1985, Zyusohonmachi, Yodogawa-ku,Osaka-shi, 532-0024 JAPAN. The “IFO strains” are available to anyperson, if desired. Bacillus stearothermophilus JTS 859 is underinternational deposit (the deposit number thereof is FERM BP-6885) inPatent Microorganism Depository, National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology.

The microorganism Bacillus stearothermophilus JTS 859 (FERM BP-6885) wasdeposited as a national deposit, on Oct. 20, 1987, in the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Japan (1-3, Higashi 1-Chome, Tsukubashi,Ibaraki-ken, Japan), which is an international deposit authoritydesignated by the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OFTHE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE.National deposit number P-9666 was assigned to the strain JTS 859.Thereafter, the strain JTS 859 was transferred from the national depositto an international deposit on Sep. 22, 1999, and international depositnumber FERM BP-6885 was assigned to it.

These types of microorganisms are grown on the normal culture medium forbacteria, and show better growth and produce a larger amount of theaforementioned enzyme by adding tryptone, yeast extract, glucose and thelike. Further, addition of a nucleic acid compound such as inosine tothe culture medium is effective in enhancing the activity of the enzyme.The cultured bacteria itself may be utilized as the crude enzyme.Alternatively, enzyme is obtained from the cultured bacteria by thestandard methods (such as destruction by supersonication or milling,centrifugation, ammonium sulfate-fractionation, and membraneseparation), and the resultant crude enzyme may also be employed.

The results of studying the bacteriological characteristics of thedeposited strain according to “Bergey's Manual of DeterminativeBacteriology, Volume II” (1984) are as follows. The experiments werebasically carried out by the method described in “Biseibutsu no Bunruito Dotei (Classification and Determination of Microorganism)” by TakejiHasegawa, edited version, Gakkai Shuppan center, 1985.

Bacillus stearothermophilus JTS 859 (FERM BP-6885)

1. Morphological Characteristics

(1) Shape and size of the cell: Rod, 5.4-6.5 μm×0.7-0.9 μm

(2) Spore: Ellipsoidal-shaped spore is formed. One spore in one cell,the spore being positioned at the end of the cell.

2. Characteristics in Culture

(1) Bouillon liquid culture: culture at 62° C. for 2 days

(2) Bouillon agar plate culture: White with slightly yellow tint,glossy, opaque, not piling up. The colony has a wavy circular shape.

(3) Bouillon agar slant culture: White with slightly yellow tint,glossy, opaque, not piling up. Moderate growth.

3. Biochemical Characteristics

 (1) Gram's stain positive  (2) Anaerobic culture no growth  (3)Motility peritrichous movement  (4) Oxidase positive  (5) Catalasepositive  (6) Liquefiability of gelatin liquefiable  (7) Litmus milkcoagulated  (8) O-F Test fermentation type  (9) VP Test negative (10)Gas generation from D-glucose no generation (11) Production of Acid fromD-glucose produced (12) Production of Acid from L-arabinose not produced(13) Production of Acid from D-mannitol produced (14) Production of Acidfrom D-xylose not produced (15) Growth in Sabouraud's medium SlantGrowth was observed Liquid Growth was observed (16) Growth under 0.001%lysozyme no growth (17) Growth under 0.02% azide no growth (18) Growthunder 7% NaCl no growth (grew up to 2% level) (19) Hydrolysis of caseinhydrolyzable (20) Hydrolysis of starch hydrolyzable (21) Egg-yolk Testno growth (22) Production of dihydroxyacetone not produced (23)Utilization of citric acid negative (24) Production of indole negative(25) Activity of urease positive (26) Deamination of phenylalaninenegative (27) Activity of arginine dihydrolase positive (28)Decomposition of tyrosine negative (29) Production of levan negative(30) Reduction of a nitrate positive (31) Denitrification of sodiumnitrate negative (32) Production of hydrogen sulfide positive (33)Utilization of inorganic nitrogen source with NO₃ as the only Growth wasobserved nitrogen source with NH₄ as the only Growth was observednitrogen source (34) GC content 47.3% (35) Growth temperature Range40-72° C. Optimum Range 60-68° C. (36) Growth pH Range 5.7-8.5 OptimumRange 6.0-7.0

Judging from the aforementioned characteristics, the present strain canbe determined to be Bacillus stearothermophilus.

<One-pot Reaction>

The method of preparing a glyoxal-guanosine-group compound and themethod of preparing a guanosine-group compound of the present inventioncan be both carried out by a one-pot reaction. Specifically, the initialreaction solution is first prepared by reacting guanosine with a glyoxalaqueous solution. The ribose-group donor (preferably together with astabilizer), the enzyme solution required for the reaction, andoptionally the phosphate ion are added, for the reaction, to the initialreaction solution, whereby a glyoxal-guanosine-group compound can beprepared by a one-pot reaction. In addition, by alkali-decomposing thereaction product, a guanosine-group compound can be prepared by aone-pot reaction.

EXAMPLES

The present invention will further be described in detail by followingProduction Examples, Experiment Example, and Examples.

Production Example 1 Synthesis of Glyoxal-guanine

22.65 g of guanine (manufactured by Tokyo Kasei Kogyo Co.,) and 21.75 gof a 40% glyoxal aqueous solution (manufactured by Tokyo Kasei KogyoCo.,) were added to 950 mL of purified water, and the mixture wasstirred with heating at 70° C. for 18 hours. The mixture was then cooledto 40° C. and filtered. The white solid obtained by the filtration wasdried, thereby resulting in 30.7 g of glyoxal-guanine. The measurementresult of nuclear magnetic resonance (NMR) spectrum is as follows.

1H-NMR (DMSO-d6, 200 MHz) δ8.52 (bs, 1H), 7.70 (bs, 1H), 7.16 (d, 1H),6.39 (d, 1H), 5.46 (d, 1H), 4.83 (d, 1H).

Production Example 2 Preparation of Bacteria Suspension which ContainNucleoside Phosphorylase

Bacillus stearothermophilus JTS 859 (FERM BP-6885), which producespurine nucleoside phosphorylase (which will be referred to as “PUNP”hereinafter) and pyrimidine nucleoside phosphorylase (which will bereferred to as “PYNP” hereinafter), was cultured in accordance with thefollowing procedure.

In culturing the bacteria, a culture medium of pH 6.2 made of 10 g ofbacto-tryptone, 5 g of yeast extract, 3 g of glucose, 3 g of salt, 1 gof inosine and 1 L of water was used. 100 mL of the aforementionedculture medium was charged into a 500 mL Erlenmeyer flask, a platinumloop of the bacteria collected from slant culture was inoculated intothe medium in the flask, and the bacteria in the flask were culturedwhile shaking gyratingly (200 rpm) at 65° C. for 16 hours, therebyresulting in cultured solution. 1.2 L of the same culture medium wascharged into a 2 L-culturing-tank, and 60 mL of the cultured solutionwas inoculated into the medium in the tank. Then, the bacteria in thetank were cultured, while two stirring vanes (the upper vane and thelower vane, each having diameter of 42 mm) were rotated at 650 rpm, for6 hours at the air flow rate of 1 vvm, at the culture temperature of 65°C. and within the pH range of 6.5-7.0 (stat). After completing theculture, the bacteria were obtained by centrifuging (10,000 G, 4° C., 15minutes). The bacteria as a whole were suspended in 40 mL of 10 mMpotassium phosphate solution (pH 7), whereby enzyme solution containingPUNP and PYNP (which will be referred to as “the enzyme solution”hereinafter) was obtained.

The enzyme activity required for converting 1 μmol of a substrate into aproduct in one minute is defined as “1U”. The enzyme activity of theenzyme solution was measured by the standard method at 60° C. 1 mL ofthe enzyme solution exhibited the PUNP activity of 66 U and the PYNPactivity of 166 U.

EXPERIMENT EXAMPLE 1 Solubility of Guanine and Glyoxal-guanine

An excess amount of each compound was suspended in water, stirred for 1hour at each temperature and left for a while. The liquid layer wasanalyzed with HPLC equipped with a UV detector, under the conditionsdescribed below. The results are shown in Table 1.

Conditions of the HPLC analysis:

Detector: UV (260 nm)

Column: Superiorex ODS (manufactured by Shiseido)

Eluent: 0.1M ammonium dihydrogenphosphate solution: methanol=97:3 (v/v)

Flow rate: 1 mL/min

Sample: 20 μL

TABLE 1 Solubility at each temperature (mg/kg) Compound 30° C. 40° C.50° C. 60° C. Guanine 2 or 2 or  2  2 less less Glyoxal-guanine 200 275330 440

Example 1 Synthesis of Glyoxal-2′-deoxyguanosine by NucleosidePhosphorylase

5 mM phosphate buffer (pH 7) containing 30 mM 2′-deoxyuridine, 30 mMglyoxal-guanine, 100 mM boric acid, and 4 mM disodiumethylenediaminetetraacetate was prepared. 1 mL of the enzyme solutionobtained in Production Example 2 was added to 99 mL of the abovephosphate buffer mixture, so that the total amount of the resultantreaction mixture was 100 mL. The reaction mixture was kept at 50° C. for48 hours, allowing the reaction to proceed. The mixture was thenfiltered at 60° C. and cooled to 4° C. The white crystal obtained by thefiltration (the yield was 80%) was subjected to spectrometry. Theresults are as follows. As a result of comparison with the valuesdescribed in the reference (Chung, F., Hecht, S. S., Carcinogenesis, 6,1671-1673 (1985)), it was confirmed that the product wasglyoxal-2′-deoxyguanosine.

UV (pH 7), λmax (nm) 248, 272 (shoulder) 1H-NMR (DMSO-d6+D20, 200 MHz),δ7.95 (s, 1H, 2-H), 6.12 (t, 1H, 1′-H), 5.50 (s, 1H, 7-H), 4.90 (s, 1H,6-H), 4.35 (bs, 1H, 31-H), 3.83 (bs, 1H, 4′-H), 3.54 (m, 2H, 5′-H), 2.52(m, 1H, 2′-H), 2.28 (m, 2H, 2′-H).

Example 2 Synthesis of 2′-deoxyguanosine from Glyoxal-2′-deoxyguanosine

0.1 g of glyoxal-2′-deoxyguanosine obtained in Example 1 described abovewas added to 0.2 N sodium hydroxide solution, and stirred at 70° C. for2 hours. After neutralizing the mixture, an analysis was performed byusing the HPLC method, under the conditions of Example 1, therebyconfirming that 2′-deoxyguanosine was quantitatively obtained. Inaddition, the white crystal obtained by chromatography andcrystallization was subjected to spectrometry, and it was confirmed fromeach spectrum that the obtained crystal was 2′-deoxyguanosine.

Example 3 The Effect of the Stabilizer on Amount ofGlyoxal-2′-deoxyguanosine Formed

5 mM phosphate buffer (pH 7) containing 20 mM 2′-deoxyuridine, 20 mMglyoxal-guanine, the stabilizer shown in Table 2 and 1 mL of the enzymesolution prepared in Production Example 2 was prepared (In Table 2, “*”indicates that the concentration of the stabilizer is 4 mM and theabsence of “*” indicates that the concentration of the stabilizer is 100mM). 100 mL of the above phosphate buffer mixture was kept at 50° C. for38 hours, allowing the reaction to proceed. Glyoxal-2′-deoxyguanosinecontained in the reaction solution was decomposed by alkali, toquantitatively obtain 2′-deoxyguanosine. Then, the concentration of2′-deoxyguanosine was determined. Further, control solution was preparedin a manner similar to that of preparing the above buffer mixture exceptfor adding 100 mM tris (hydroxymethyl) aminomethane hydrochloric acid(“Tris”) instead of the stabilizer. This solution will be referred to as“Tris (control)” hereinafter. From the analysis on the results, it wasdiscovered that the combination of a complex-forming-compound similar toEDTA with boric acid is effective for improving the reaction yield.

TABLE 2 Amount of glyoxal-2′- deoxyguanosine Stabilizer formed (mM) Tris(control) 5.4 Boric acid 4.0 Glycine 6.0 Iminodiacetic acid * 8.8Nitrilotriacetic acid * 5.4 EDTA * 9.5 EGTA * 8.0 Tris and EDTA * 9.8Glycine and EDTA * 10.1 Boric acid and Tris 5.6 Boric acid and glycine6.5 Boric acid and iminodiacetic acid * 12.4 Boric acid andnitrilotriacetic acid * 12.2 Boric acid and EDTA * 12.8 Boric acid andEGTA * 11.5

Example 4 Synthesis of Glyoxal-2′-deoxyguanosine by the Enzyme Derivedfrom Each Strain

500 mL of the same culture medium as used in Production Example 2 wascharged into each of three 2 L Erlenmeyer flasks. Escherichia coli IFO3301, Escherichia coli IFO 13168 and Klebsiella pneumoniae IFO 3321 waseach inoculated into the medium in each of three flasks, by collecting aplatinum loop of bacteria each from the stock slant culture. The abovebacteria each were cultured while shaking the flasks (200 rpm) at 37° C.for 16 hours, thereby resulting in culture solution. The bacteria werecollected by centrifuging (10,000 G, 4° C., 15 minutes) the culturesolution. The obtained bacteria was suspended in 10 mL of 10 mMpotassium phosphate buffer (pH 7), thereby resulting in three sets ofenzyme solutions.

Glyoxal-2′-deoxyguanosine was synthesized from 2′-deoxyuridine andglyoxal-guanine under the following conditions, by using any one of theaforementioned three sets of enzyme solutions and the Bacillusstearothermophilus JTS 859 enzyme solution obtained in ProductionExample 2. Specifically, 5 mM phosphate buffer (pH 7) containing 30 mM2′-deoxyuridine, 30 mM glyoxal-guanine, 100 mM boric acid, and 4 mMdisodium ethylenediaminetetraacetate was first prepared. Each (2 mL) ofthe above four sets of enzyme solutions (which contain bacteriacorresponding to 100 mL of the above culture solution) was added to 98mL of the above phosphate buffer mixture, respectively, resulting infour sets of reaction mixtures. Each reaction mixture was kept at eachreaction temperature for 48 hours, allowing the reaction to proceed. Theresults are shown in Table 3.

TABLE 3 Yield of Reaction glyoxal-2′- temperature deoxyguanosine Strain(° C.) (%) Bacillus 50 79 stearothermophilus JTS 859 Escherichia coli 3017 IFO 3301 Escherichia coli 30 40 IFO 13168 Klebsiella pneumoniae 30 23IFO 3321

Example 5 Synthesis of a Glyoxal-guanosine-group Compound by UsingVarious Ribose-group Donor

5 mM phosphate buffer (pH 7) containing each ribose-group donor as shownin Table 4, 20 mM glyoxal-guanine, 100 mM boric acid, and 4 mM disodiumethylenediaminetetraacetate was first prepared (the concentration ofeach ribose-group donor was 20 mM. However, “*” in Table 4 indicatesthat the concentration was 10 mM). 0.25 mL of the enzyme solutionobtained in Production Example 2 was added to a required amount of theabove phosphate buffer mixture, so that the total amount of theresultant reaction mixture was 10 mL. The reaction mixture was kept at50° C. for 48 hours, allowing the reaction to proceed. The results areshown in Table 4.

TABLE 4 Yield Ribose-group donor Nucleoside formed (%)Ribose-1-phosphate * Glyoxal-guanosine 78 2-Deoxyribose-1- Glyoxal-2′-76 phosphate * deoxyguanosine Uridine Glyoxal-guanosine 612′-Deoxyuridine Glyoxal-2′- 79 deoxyguanosine Thymidine Glyoxal-2′- 63deoxyguanosine

Example 6 Effect of the Substrate Concentration on the Preparation Usingthe Enzyme Derived from Bacillus stearothermophilus JTS 859

5 mM phosphate buffer (pH 7) containing guanine or glyoxal-guanine asthe substrate base of each concentration shown in Table 5,2′-deoxyuridine as the ribose-group donor having the same concentrationas each substrate base, 100 mM boric acid, and 4 mM disodiumethylenediaminetetraacetate was first prepared. 1 mL of the enzymesolution obtained in Production Example 2 was added to 99 mL of theabove phosphate buffer mixture. The resultant reaction mixture was keptat 50° C. for 48 hours, allowing the reaction to proceed. The resultsare shown in Table 5. When glyoxal-guanine was selected as the substratebase, the formed glyoxal-2′-deoxyguanosine was decomposed by alkali, toobtain 2′-deoxyguanosine. The amount of the obtained 2′-deoxyguanosinewas determined. In Table 5, the analysis was not performed when theconcentration of guanine was no less than 50 mM.

TABLE 5 Concentration Yield of 2′-deoxyguanosine (%) of substrate 10 2030 50 70 80 100 base (mM) mM mM mM mM mM mM mM Guanine 29 23 19 — — — —(Comparative example) Glyoxal-guanine 81 79 79 77 72 66 64

Example 7 Synthesis by One-pot Reaction

0.45 g of guanosine and 0.44 g of a 40% glyoxal aqueous solution wereadded to 10 mL of water, and the mixture was stirred at 70° C. for 18hours. Next, the following substances and water were added to the abovemixture, thereby resulting in the aqueous solution containing 30 mM2′-deoxyuridine, 100 mM boric acid, 4 mM disodiumethylenediaminetetraacetate, and 5 mM potassium phosphate, as finalconcentration. 1 mL of the enzyme solution obtained in ProductionExample 2 was added to 99 mL of the above aqueous solution, so that thetotal volume of the resultant reaction mixture was 100 mL. The reactionmixture was kept at 50° C. for 48 hours, allowing the reaction toproceed. Glyoxal-2′-deoxyguanosine contained in the reaction mixture wasdecomposed by alkali, to 2′-deoxyguanosine. Measurement of theconcentration of 2′-deoxyguanosine by HPLC revealed that the yield was80%.

Example 8 Synthesis of Glyoxal-guanosine

5 mM phosphate buffer (pH 7) containing 50 mM uridine, 50 mMglyoxal-guanine, 100 mM boric acid, and 4 mM disodiumethylenediaminetetraacetate was first prepared. 1 mL of the enzymesolution obtained in Production Example 2 was added to 99 mL of theabove phosphate buffer mixture, so that the total volume of theresultant reaction mixture was 100 mL. The reaction mixture was kept at50° C. for 48 hours, allowing the reaction to proceed. After beingfiltered with heating, the mixture was cooled to 4° C. The white crystalobtained by the filtration (the yield was 72%) was subjected tospectrometry. The results are as follows. As a result of comparison withspectrum of glyoxal-2′-deoxyguanosine of Example 1, it was confirmedthat the product was glyoxal-guanosine.

1H-NMR (DMSO-d6, 200 MHz), δ8.85 (s, 1H, 5-H), 8.00 (s, 1H, 2-H), 7.27(d, 1H, 7-OH), 6.50 (d, 1H, 6-OH), 5.71 (d, 1H, 1′-H), 5.49 (d, 1H,7-H), 5.44 (d, 1H, 3′-OH), 5.18 (d, 1H, 2′-OH), 5.06 (s, 1H, 5′-OH),4.88 (d, 1H, 6-H), 4.41 (m, 1H, 2′-H), 4.10 (d, 1H, 3′-H), 3.90 (d, 1H,4′-H), 3.57 (m, 2H, 5′-H).

Further, guanosine was prepared from the product obtained in thisExample, in accordance with the procedure similar to that of Example 2.This fact also revealed that the product obtained in this Example wasglyoxal-guanosine.

According to the present invention, a glyoxal-guanosine-group compoundcan be prepared from the ribose-group donor and the glyoxal-guanine, bythe enzyme reactions derived from the microorganism. By alkalidecomposing the glyoxal-guanosine-group compound, a guanosine-groupcompound consisting of guanosine and 2′-deoxyguanosine can beefficiently prepared at a high yield.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

What is claimed is:
 1. A method of preparing a glyoxal-guanosine-groupcompound represented by the formula (2):

wherein R represents a hydrogen atom or a hydroxyl group, whichcomprises the step of; reacting glyoxal-guanine represented by theformula (1):

with ribose-1-phosphate or 2-deoxyribose-1-phosphate with a microbialpurine nucleoside phosphorylase, thereby obtaining a glyoxal-guanosineor glyoxal-2′-deoxyguanosine.
 2. The method of preparing aglyoxal-guanosine-group compound according to claim 1, wherein, themicrobial purine nucleoside phosphorylase, is contained in amicroorganism or said enzyme is obtained from a microorganism.
 3. Themethod of preparing a glyoxal-guanosine-group compound according toclaim 2, wherein the microorganism belongs to Bacillus genus,Escherichia genus or Klebsiella genus.
 4. The method of preparing aglyoxal-guanosine-group compound according to claim 3, wherein themicroorganism is Bacillus stearothermophilus JTS 859 (FERM BP-6885),Escherichia coli IFO 3301, Escherichia coli IFO 13168, or Klebsiellapneumoniae IFO
 3321. 5. The method of preparing aglyoxal-guanosine-group compound according to claim 4, wherein at leastone compound selected from the group consisting of glycine,iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraaceticacid, ethylene glycol bis(β-aminoethyl ether) -N,N,N′,N′-tetraaceticacid and salts thereof is added, or the above at least one compound isadded in combination with boric acid or a salt thereof.
 6. The method ofpreparing a glyoxal-guanosine-group compound according to claim 3,wherein at least one compound selected from the group consisting ofglycine, iminodiacetic acid, nitrilotriacetic acid,ethylenediaminetetraacetic acid, ethylene glycol bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid and salts thereof is added, or theabove at least one compound is added in combination with boric acid or asalt thereof.
 7. The method of preparing a glyoxal-guanosine-groupcompound according to claim 2, wherein the microorganism is Bacillusstearothermophilus JTS 859 (FERM BP-6885), Escherichia coli IFO 3301,Escherichia coli IFO 13168, or Kliebsiella pneumoniae IFO
 3321. 8. Themethod of preparing a glyoxal-guanosine-group compound according toclaim 7, wherein at least one compound selected from the groupconsisting of glycine, iminodiacetic acid, nitrilotriacetic acid,ethylenediaminetetraacetic acid, ethylene glycol bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid and salts thereof is added, or theabove at least one compound is added in combination with boric acid or asalt thereof.
 9. The method of preparing a glyoxal-guanosine-groupcompound according to claim 2, wherein at least one compound selectedfrom the group consisting of glycine, iminodiacetic acid,nitrilotriacetic acid, ethylenediaminetetraacetic acid, ethylene glycolbis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid and salts thereof isadded, or the above at least one compound is added in combination withboric acid or a salt thereof.
 10. The method of preparing aglyoxal-guanosine-group compound according to claim 1, wherein at leastone compound selected from the group consisting of glycine,iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraaceticacid, ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acidand salts thereof is added, or the above at least one compound is addedin combination with boric acid or a salt thereof.